Anne Rousseau

Separation of propranolol enantiomers by CE using sulfated beta-CD derivatives in aqueous and non-aqueous electrolytes: Comparative CE and NMR study.

Separations using CE employing non‐aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with non‐aqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non‐aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non‐aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non‐aqueous methanolic BGEs with two single isomer sulfated derivatives of β‐CD, namely heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD and heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non‐aqueous BGE in the case of heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD but remained the same in the case of heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non‐aqueous BGEs.

Capillary electrophoretic and nuclear magnetic resonance studies on the opposite affinity pattern of propranolol enantiomers towards various cyclodextrins

In the present study the migration order of the propranolol enantiomers with various native CDs and neutral and charged CD derivatives was examined in capillary electrophoresis (CE). The reversal of the enantiomer migration order was observed due to sulfation of beta-CD on its primary hydroxy groups. The structures of intermolecular selector-select and temporary diastereomeric associates in solution were elucidated based on 1D rotating frame nuclear Overhauser effect spectroscopy (1D ROESY) experiments. Major structural differences were observed between the propranolol complexes with native beta-CD and heptakis(6-O-sulfo)-beta-CD.

Influence of the BGE composition on analyte response in CD‐mediated NACE‐MS

The influence of the BGE composition, including the addition of a single‐isomer sulfated β‐CD derivative, on the ionization performance of the model compound carvedilol in NACE‐ESI‐MS was studied using an alternative infusion method. This approach employs voltage‐induced infusion of the BGE containing the analyte, and takes into account the effects of variations in EOF and effective analyte mobility on the ESI‐MS intensity. First, the optimal composition of the sheath liquid for CE‐MS in terms of signal abundance and stability was determined. The BGE ammonium formate, acetate, and camphorsulfonate were found to have similar effects on analyte ionization. Addition of single‐isomer sulfated β‐CD derivatives (available as sodium salt) to the BGE revealed that the anionic CD derivatives did not give rise to the same ionization suppression effect. This result can be attributed to differences in the dissociation state of these sodium salts. Finally, it is shown that information about chiral selectivity can also be obtained with the applied infusion method.

Combination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged β-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-β-CD or HDAS-β-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained.

Development and validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans using a single-isomer anionic cyclodextrin derivative and an ionic liquid

The enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans, i.e. 4-amino-2,2-dimethyl-6-ethoxycarbonylamino-3,4-dihydro-2H-1-benzopyran, was successfully carried out using an anionic cyclodextrin (CD) derivative combined with a chiral ionic liquid (IL). In order to obtain high resolution and efficiency values, the addition of a chiral IL, i.e. ethylcholine bis(trifluoromethylsulfonyl)imide (EtChol NTf(2)), to the background electrolyte containing heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) was found to be essential. A simultaneous increase in separation selectivity and enantioresolution seems to indicate a synergistic effect of HDMS-β-CD and EtChol NTf(2). The best enantioseparation of the key intermediate was achieved using a methanolic solution of 0.75M formic acid, 10mM ammonium formate, 1.5mM HDMS-β-CD and 5mM EtChol NTf(2). Levamisole was selected as internal standard. The optimized conditions allowed the determination of 0.1% of each enantiomer in the presence of its stereoisomer using the method of standard additions. The NACE method was then fully validated with respect to selectivity, response function, trueness, precision, accuracy, linearity and limits of detection and quantification.

Validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of R-flurbiprofen using a single-isomer amino cyclodextrin derivative

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of R-flurbiprofen using 6-monodeoxy-6-mono(2-hydroxy)propylamino-beta-cyclodextrin (IPA-beta-CD) as chiral selector. The nonaqueous BGE was made up of 20 mM IPA-beta-CD, 20 mM ammonium camphorsulfonate and 40 mM ammonium acetate in methanol. Flufenamic acid was selected as internal standard. The CE method was carefully optimized in order to prevent the adsorption of the cationic CD onto the capillary wall, and therefore, to avoid loss of peak efficiency and enantioresolution. To achieve this goal, the addition of ammonium camphorsulfonate was found to be necessary. In the selected conditions, the determination of 0.1% of S-flurbiprofen in R-flurbiprofen could be performed using the method of standard additions. The NACE method was then fully validated by applying a novel strategy using accuracy profiles.

Determination of flurbiprofen enantiomers in plasma using a single‐isomer amino cyclodextrin derivative in nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) assay was developed for the separation and determination of flurbiprofen enantiomers in plasma samples using 6‐monodeoxy‐6‐mono(3‐hydroxy)propylamino‐β‐cyclodextrin as chiral selector. The nonaqueous background electrolyte was made up of 40 mM ammonium acetate in methanol (MeOH), and flufenamic acid was employed as internal standard. Solid‐phase extraction was used for sample cleanup prior to the NACE separation.

Effect of the nature of the single-isomer anionic CD and the BGE composition on the enantiomeric separation of β-blockers in NACE

The separation of ten β‐blockers has been investigated in NACE systems using heptakis(2,3‐di‐O‐methyl‐6‐O‐sulfo)‐β‐CD (HDMS‐β‐CD) and heptakis(2,3‐di‐O‐acetyl‐6‐O‐sulfo)‐β‐CD (HDAS‐β‐CD). The influence on enantioresolution, mobility difference and selectivity of the nature of both anionic CD and BGE anion as well as their concentrations were studied by means of a multivariate approach. A D‐optimal design with 25 experimental points was applied. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by both CD nature and concentration. Except for two compounds, HDAS‐β‐CD was found to give higher enantioresolution values than HDMS‐β‐CD. The best enantioseparation for all compounds was achieved in the presence of a high chiral selector concentration and for most of them at a low BGE anion concentration. For each investigated compound, operating conditions leading to the best enantiomeric resolution were deduced. A generic NACE system was then recommended, namely 10 mM ammonium acetate and 40 mM HDAS‐β‐CD in methanol acidified with 0.75 M formic acid. This generic system was able to completely resolve the enantiomers of all β‐blockers, with a Rs value of at least 4. Finally, the optimal conditions obtained modelling resolution, mobility difference and selectivity were compared.

Generic systems for the enantioseparation of basic drugs in NACE using single-isomer anionic CDs

The enantioseparation of 10 basic drugs was evaluated in NACE systems using heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-β-CD (HMAS-β-CD). For this purpose, a D-optimal design with 21 experimental points was applied. Four antifungal agents (econazole, isoconazole, miconazole, sulconazole), three local anesthetics (bupivacaine, mepivacaine and prilocaine), two sympathomimetics (salbutamol and terbutaline) and one β-blocker (carvedilol) were selected as basic model analytes. The influence on the enantiomeric resolution of anionic CD and BGE anion concentrations as well as the BGE anion nature was investigated. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by the CD concentration. Based on the observed results, a generic NACE system was recommended, namely 20mM HMAS-β-CD and 10mM ammonium camphor SO(3)(-) in methanol acidified with 0.75 M formic acid. Moreover, this NACE system was compared to previous conditions with heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) or heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD). Finally, two generic systems using either HDAS-β-CD or HMAS-β-CD were proposed and evaluated for the enantioseparation of ketamine and norketamine after incubation of ketamine in phenobarbital-induced male rat liver microsomes systems.

Association of two single-isomer anionic CD in NACE for the chiral and achiral separation of fenbendazole, its sulphoxide and sulphone metabolites: application to their determination after in vitro metabolism.

A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and nonchiral (FBZ sulphone or FBZSO2) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3‐di‐O‐acetyl‐6‐O‐sulfo)‐β‐CD and 10 mM heptakis(2,3‐di‐O‐methyl‐6‐O‐sulfo)‐β‐CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ, OFZ enantiomers, and FBZSO2. Thiabendazole was selected as an internal standard. The CD‐NACE potential was then evaluated for in vitro metabolism studies using FBZ as a model case. The OFZ enantiomers and FBZSO2 could be detected after incubation of FBZ in the phenobarbital‐induced male rat liver microsomes systems.